The overall goal of this project is to understand how telomeres prevent the activation of the DNA damage response at chromosome ends. In the prior funding period we have made progress on defining this end- protection problem, establishing that the two main DNA damage response pathways, the ATM and ATR kinase signaling cascades, can be activated at chromosome ends when telomere protection is lost. In this proposal, we describe experiments designed to gain mechanistic insights into how telomeres repress the ATM and ATR kinases. Specifically, AIMs 1 and 2 will focus on two components of the telomeric shelterin complex, TRF2 and POT1, which we have shown to act independently to repress the ATM and ATR pathways at chromosome ends. The third aim of this proposal is to examine how telomeres are duplicated by semi-conservative DNA replication. Our preliminary data indicates that telomeric repeats pose a considerable challenge to the replication fork, leading to replication fork arrest and phenotypes consistent with telomeres representing fragile sites. Thus, the data suggest that the protective telomeric DNA at chromosome ends comes at the cost of potential replication problems. TRF1, a third component of shelterin identified in our laboratory, was found to have a key role in facilitating the replication of telomeres. This new insight forms the basis of the work in AIM 3 where we will examine telomere replication in detail and address how TRF1 facilitates replication fork progression through the telomeric DNA. Together these experiments should reveal the basic principles by which the telomeric shelterin complex allows mammalian cells to maintain linear chromosomes without activating DNA damage signaling cascades. Human cells lose telomeric DNA with cell divisions and exhibit limited proliferation due to the eventual loss of telomere function. Understanding telomere function and the consequences of telomere deprotection is directly relevant to the limited life-span of primary human cells in vitro and will shed light on the finite ability of stem cells to replenish organ function in vivo. This research is therefore expected to improve the understanding of telomere- related diseases, such as dyskeratosis congenita, and diminished organ function with aging in normal individuals. Furthermore, the outcome of our experiments are relevant to human cancer since loss of telomere function is thought to be a driving force in the early stages of human tumorigenesis.